Specificity of MCD treatment. (A) Splenic T cells were treated with 10 mM MCD (filled histogram) or not (open histogram with solid line), and cholesterol in the plasma membrane was stained with 12.5 μg/mL filipin and analyzed by flow cytometer. The open histogram with broken line shows autofluorescence of unstained splenic T cells. (B) Splenic T cells were treated with 10 mM MCD, and after washing away MCD, they were incubated with water-soluble cholesterol (60 μg/mL) at 37°C for 30 minutes for cholesterol reconstitution. The treated cells were analyzed for LFA-1–mediated adhesion to ICAM-1 as in Figure 1A. Open bar (□) indicates unstimulated cells; solid bar (▪), PMA stimulation; gray bar (▦), PMA stimulation and anti–LFA-1 antibody blocking; horizontally striped bar (▤), 10 mM MCD treatment after PMA stimulation; and diagonally striped bar (▨), PMA stimulation and MCD treatment followed by cholesterol reconstitution. Error bars indicate SD. (C) Splenic T cells were stimulated with (+) or without (-) PMA in the presence (+) or absence (-) of MCD and the phosphorylation of MAP kinase was analyzed by Western blotting using anti–phospho-ERK-1/2 antibody (top panel). The same blot was stripped and probed with anti–ERK-1/2 antibody to confirm equal loading of the samples (bottom panel).